Direct in vivo cell lineage analysis in the retrorsine and 2AAF models of liver injury after genetic labeling in adult and newborn rats.

Pichard V, Aubert D, Ferry N - PLoS ONE (2009)

Bottom Line:
Retrorsine treatment did not modify the overall number of labeled cells in the liver whereas after 2-AAF administration unlabeled oval cells were recorded and the total number of labeled cells decreased significantly.In contrast, in the 2-AAF regimen numerous labeled oval cells were present and were able to generate new labeled hepatocytes.Our results strongly suggest that SHPCs are derived from hepatocytes and we confirm that SHPCs and oval cells do not share the same origin.

Backgrounds and aims: When hepatocyte proliferation is impaired, liver regeneration proceeds from the division of non parenchymal hepatocyte progenitors. Oval cells and Small Hepatocyte-like Progenitor Cells (SHPCs) represent the two most studied examples of such epithelial cells with putative stem cell capacity. In the present study we wished to compare the origin of SHPCs proliferating after retrorsine administration to the one of oval cells observed after 2-Acetyl-Amino fluorene (2-AAF) treatment.

Methodology/principal findings: We used retroviral-mediated nlslacZ genetic labeling of dividing cells to study the fate of cells in the liver. Labeling was performed either in adult rats before treatment or in newborn animals. Labeled cells were identified and characterised by immunohistochemistry. In adult-labeled animals, labeling was restricted to mature hepatocytes. Retrorsine treatment did not modify the overall number of labeled cells in the liver whereas after 2-AAF administration unlabeled oval cells were recorded and the total number of labeled cells decreased significantly. When labeling was performed in newborn rats, results after retrorsine administration were identical to those obtained in adult-labeled rats. In contrast, in the 2-AAF regimen numerous labeled oval cells were present and were able to generate new labeled hepatocytes. Furthermore, we also observed labeled biliary tracts in 2-AAF treated rats.

Conclusions: Our results strongly suggest that SHPCs are derived from hepatocytes and we confirm that SHPCs and oval cells do not share the same origin. We also show that hepatic progenitors are labeled in newborn rats suggesting future directions for in vivo lineage studies.

Mentions:
In the retrorsine group, the liver lobes removed at partial hepatectomy revealed a mean proportion of 6.3±3% of β-galactosidase positive hepatocytes (Table 1). This value is in the same range than in animals that did not receive retrorsine [11], [12]. We also observed, that cells other than hepatocytes were not labeled (<0.01%) as previously described [12]. The labeled cells were randomly distributed in the liver lobule, indicating indiscriminate infection of hepatocytes with the retroviral vector [11], [20], [21]. In two animals (R4 and R6 in Table 1), no β-galactosidase positive hepatocytes were found. However, anti β-galactosidase antibodies were found in the serum indicating that some animals may escape immune suppression induced by CTLA4Ig. Four animals were sacrificed at day 15 after hepatectomy. In these animals the mean proportion of β-galactosidase positive hepatocytes was 4.7±1.5% (Figure 2). Moreover, we observed the presence of clusters of small-size hepatocytes (less than 20 cells in size) which corresponded to Small Hepatic Progenitor cells (SHPCs) as previously described [10]. These clusters were highly labeled with anti Ki67 antibody indicating a high proliferation index. As shown in Figure 3A some clusters also expressed β-galactosidase and the proportion of clusters expressing β-galactosidase was similar to the proportion of hepatocytes that were initially labeled (Table 1). Four animals were sacrificed at 4 weeks after hepatectomy. In these animals, we found larger clusters of small hepatocytes, resulting from the division of SHPCs as expected in the retrorsine model at this time point [10], [11]. Moreover, at that time the Ki67 labeling was much lower, indicating a drop in cell proliferation in the clusters. We also observed that some clusters expressed β-galactosidase (Figure 3B). Because these large clusters frequently merged, precise quantification of the proportion of β-glactosidse positive clusters was not possible. However, the mean overall proportion of β–gal positive hepatocytes was 4.6±0.7%. We never detected clear oval cell proliferation in any section at sacrifice although transient ductular reaction was sometimes seen. Cytokeratin staining was limited to biliary epithelial cells. These data recapitulated our previous results and showed that the mean proportion of β-galactosidase positive hepatocytes was not statistically different in livers harvested at hepatectomy or at the two times of sacrifice (p = 0.4 using ANOVA). Moreover, the proportion of β-galactosidase clusters at day 15 was identical to the proportion of labeled hepatocytes, strongly suggesting that the clusters originated from the labeled hepatocytes that were present in the liver before retrorsine administration. To confirm that the presence of merging clusters of various sizes did not impact the counting of hepatocytes, we verified that β-galactosidase enzymatic activity was identical in liver homogenates from rats sacrificed at day 15 or day 30 (17.9±6.1 ng βgal/mg protein at day 15 vs 18.5±5.6 at day 30; p = 0.9 using Student's t test).

Mentions:
In the retrorsine group, the liver lobes removed at partial hepatectomy revealed a mean proportion of 6.3±3% of β-galactosidase positive hepatocytes (Table 1). This value is in the same range than in animals that did not receive retrorsine [11], [12]. We also observed, that cells other than hepatocytes were not labeled (<0.01%) as previously described [12]. The labeled cells were randomly distributed in the liver lobule, indicating indiscriminate infection of hepatocytes with the retroviral vector [11], [20], [21]. In two animals (R4 and R6 in Table 1), no β-galactosidase positive hepatocytes were found. However, anti β-galactosidase antibodies were found in the serum indicating that some animals may escape immune suppression induced by CTLA4Ig. Four animals were sacrificed at day 15 after hepatectomy. In these animals the mean proportion of β-galactosidase positive hepatocytes was 4.7±1.5% (Figure 2). Moreover, we observed the presence of clusters of small-size hepatocytes (less than 20 cells in size) which corresponded to Small Hepatic Progenitor cells (SHPCs) as previously described [10]. These clusters were highly labeled with anti Ki67 antibody indicating a high proliferation index. As shown in Figure 3A some clusters also expressed β-galactosidase and the proportion of clusters expressing β-galactosidase was similar to the proportion of hepatocytes that were initially labeled (Table 1). Four animals were sacrificed at 4 weeks after hepatectomy. In these animals, we found larger clusters of small hepatocytes, resulting from the division of SHPCs as expected in the retrorsine model at this time point [10], [11]. Moreover, at that time the Ki67 labeling was much lower, indicating a drop in cell proliferation in the clusters. We also observed that some clusters expressed β-galactosidase (Figure 3B). Because these large clusters frequently merged, precise quantification of the proportion of β-glactosidse positive clusters was not possible. However, the mean overall proportion of β–gal positive hepatocytes was 4.6±0.7%. We never detected clear oval cell proliferation in any section at sacrifice although transient ductular reaction was sometimes seen. Cytokeratin staining was limited to biliary epithelial cells. These data recapitulated our previous results and showed that the mean proportion of β-galactosidase positive hepatocytes was not statistically different in livers harvested at hepatectomy or at the two times of sacrifice (p = 0.4 using ANOVA). Moreover, the proportion of β-galactosidase clusters at day 15 was identical to the proportion of labeled hepatocytes, strongly suggesting that the clusters originated from the labeled hepatocytes that were present in the liver before retrorsine administration. To confirm that the presence of merging clusters of various sizes did not impact the counting of hepatocytes, we verified that β-galactosidase enzymatic activity was identical in liver homogenates from rats sacrificed at day 15 or day 30 (17.9±6.1 ng βgal/mg protein at day 15 vs 18.5±5.6 at day 30; p = 0.9 using Student's t test).

Bottom Line:
Retrorsine treatment did not modify the overall number of labeled cells in the liver whereas after 2-AAF administration unlabeled oval cells were recorded and the total number of labeled cells decreased significantly.In contrast, in the 2-AAF regimen numerous labeled oval cells were present and were able to generate new labeled hepatocytes.Our results strongly suggest that SHPCs are derived from hepatocytes and we confirm that SHPCs and oval cells do not share the same origin.

Backgrounds and aims: When hepatocyte proliferation is impaired, liver regeneration proceeds from the division of non parenchymal hepatocyte progenitors. Oval cells and Small Hepatocyte-like Progenitor Cells (SHPCs) represent the two most studied examples of such epithelial cells with putative stem cell capacity. In the present study we wished to compare the origin of SHPCs proliferating after retrorsine administration to the one of oval cells observed after 2-Acetyl-Amino fluorene (2-AAF) treatment.

Methodology/principal findings: We used retroviral-mediated nlslacZ genetic labeling of dividing cells to study the fate of cells in the liver. Labeling was performed either in adult rats before treatment or in newborn animals. Labeled cells were identified and characterised by immunohistochemistry. In adult-labeled animals, labeling was restricted to mature hepatocytes. Retrorsine treatment did not modify the overall number of labeled cells in the liver whereas after 2-AAF administration unlabeled oval cells were recorded and the total number of labeled cells decreased significantly. When labeling was performed in newborn rats, results after retrorsine administration were identical to those obtained in adult-labeled rats. In contrast, in the 2-AAF regimen numerous labeled oval cells were present and were able to generate new labeled hepatocytes. Furthermore, we also observed labeled biliary tracts in 2-AAF treated rats.

Conclusions: Our results strongly suggest that SHPCs are derived from hepatocytes and we confirm that SHPCs and oval cells do not share the same origin. We also show that hepatic progenitors are labeled in newborn rats suggesting future directions for in vivo lineage studies.